Cloning of the zymocin gene and use of zymocin in beverages

ABSTRACT

The present invention discloses a method for the preservation of beverages comprising the use of a killer toxin. Specifically, zymocin is used in carbonated drinks. The zymocin is shown to be active against a wide range of different yeast strains. The invention further discloses a DNA fragment encoding zymocin. The DNA sequence is also disclosed.

TECHNICAL FIELD

The present invention discloses a method for preserving beverages comprising the use of a killer toxin. Specifically an effective amount of a zymocin is used. The zymocin is particularly used in carbonated drinks. The invention also discloses the cloning of the Williopsis mrakii DNA and the DNA sequence encoding zymocin. This makes possible overexpression and large scale production of zymocin.

BACKGROUND OF THE INVENTION

Spoilage of food by yeasts is a well known problem in food industry. In some products yeasts are the main source of spoilage. Fruits belong to the natural habitat of yeasts. Therefore, it is not surprising that fruit derived products and fruit containing products are susceptible to contamination by yeasts. Examples are fruit pulp, fruit juice, fruit containing softdrinks and carbonated beverages, which sometimes contain high percentages of fruit juice. Spoilage of those products is even a major problem in industry. Softdrinks and carbonated beverages are to be considered as a very selective environment for the growth of yeasts. This environment has peculiar physical characteristics specifically low pH, low oxygen concentration and high sugar concentration. Furthermore storage is at low temperature. Yeasts have the ability to ferment the sugars introduced as such or those introduced with the fruits. Outgrowth of yeasts is responsible for off-flavours, there is loss of texture quality and gas is produced. The production of gas can cause swelling or even blowing up of the container. In the case of carbonated beverages bottled in glass this phenomenon has caused severe injuries. Sellar. P. W. and P. B. Johnston (BMJ (1991) 303 176-177)) describe how the seriousness of ocular injuries caused by exploding bottles has been underestimated. T. Willhoft (New Scientist (1986) 21 Aug., 28-30) also reports the popping bottle problem. The problem is due to carbon dioxide and although not specifically reported it is known that this gas is also produced by yeasts.

Yeast genera most frequently isolated from fruit and fruit products are Saccharomyces, Kluvveromyces, Zygosaccharomyces, Debaryomyces, Hansenula, Candida, Pichia and Torulopsis. The yeasts are introduced into the products with the ingredients such as the fruits. The yeasts also may grow on the surfaces of the production equipment.

In spite of good hygienic production methods and the use of preservatives like sorbate and benzoate spoilage of fruit products is still very common. Chemical preservatives like sorbic acid and benzoic acid pose their own problems. The concentration of these chemicals needed to prevent the outgrowth of some yeast species commonly detected in fruit derived products is very high. The concentration of benzoate and/or sorbate permitted in those products is too low to prevent outgrowth of these yeasts.

Killer toxins are inhibitory molecules produced by a wide range of yeasts. The inhibitory molecules are polypeptides or polypeptide containing molecules which kill sensitive yeast cells. Killer toxins were first observed in certain strains of the genus Saccharomyces (Makower M. et al. (1963) Proc. 11th Int. Congr. Genet. I, 1202). Today many different killer toxin-producing yeast species are known (Young T. W. (1987) in The Yeasts, Rose A. H. et al. Eds. Acad. Press, London. 2: 131-164). The majority of the killer toxins are not very stable and therefore not useful as a preservative. However, various Hansenula species make an exception to this rule and produce very heat- and pH-stable killer toxins (Nomoto, H. et al. (1984) Agric. Biol. Chem. 48 : 807-809).

For commercial use as an anti-yeast preservative it is preferred to employ a stable killer toxin with a broad spectrum anti-yeast activity.

An example of such a killer toxin is the killer toxin produced and secreted by Hansenula mrakii IFO 0895. The killer toxin of Hansenula mrakii IFO 0895 is very stable against heat and in a pH range of 4-11. A broad spectrum activity was demonstrated (Ashida S (1983) Agric. Biol. Chem. 47: 2953-2955). Further characterization showed that the killer toxin is a polypeptide with a molecular mass of 10721 Da. The molecule contains 88 amino acid residues. The amino acid sequence of this polypeptide has been published (Yamamoto T. et al. (1986) FEBS 195: 253-257). The name of this microorganism has in the meantime been changed to Williopsis mrakii.

Another example of a stable killer toxin is the killer toxin produced by Hansenula saturnus. This killer toxin shows a large similarity in activity spectrum and heat stability with the Williopsis mrakii killer toxin, hereinafter further indicated as zymocin. The Hansenula saturnus killer toxin is a small polypeptide with a molecular mass of 8500 Da and an isoelectric point of 5.8 (Ohta, T. et al. (1984) Agric. Biol. Chem. 48 : 903-908).

The potential use of killer toxins--especially the above mentioned killer toxin--as a preservative against yeast spoilage in several food products is described, in Japanese patent application JP 62-40272.

The use of a killer toxin for the prevention of outgrowth of yeasts in fruit containing carbonated beverages, which are to be considered as products that are very sensitive for spoilage by yeasts, has not been reported to date.

SUMMARY OF THE INVENTION

The present invention describes a method for the preservation of beverages comprising the use of a killer toxin. The killer toxin is used in carbonated and non-carbonated beverages. The zymocin is used as such or in combination with other preservation systems. The zymocin prevents the outgrowth of a wide range of yeast species in carbonated beverages, especially fruit containing beverages.

The present invention also discloses the DNA sequence encoding zymocin obtainable from Williopsis mrakii.

The invention further discloses a DNA fragment containing the gene encoding the said zymocin.

Another aspect of the invention discloses vectors and host strains for producing zymocin.

The present invention makes possible the cloning, overexpression and large scale production of zymocins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the partial nucleotide sequence (SEQ ID NO:1) of a 5 kb PstI fragment of Williopsis mrakii DNA containing the zymocin gene and the amino acid sequence (SEQ ID NO:2) of the coding region.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes the use of killer toxins. The killer toxins can be used as such they can also be used in combination with other preservation systems. Killer toxins can also be used together with other killer toxins. They asure the prevention of outgrowth of a wide range of yeast species in carbonated beverages, especially fruit containing beverages. In general the yeasts or other species are introduced in the drinks together with the fruits. It is also possible that the contamination occurs during the processing of the fluids or the fruits. The invention is therefore not restricted to beverages containing fruit material.

The killer toxins of the present invention are obtainable for Williopsis mrakii. They are characterized by their high pH- and thermal stability. Similar killer toxins also suitable in the uses claimed in the present invention are obtainable from Hansenula saturnus. Collectively these killer toxins are indicated as zymocins.

It is shown that the zymocins of the present invention are active against yeasts in carbonated beverages, both alcoholic- and non-alcoholic. Specifically, the zymocins are active in BRON ORANGE™, JAFFA DRINK™, 7-UP™, CERISE™ and STENDER BIER™. It is also demonstrated that the zymocins are active in fruit juices such as apple juice, orange juice and pear juice and black currant drink.

As representatives of yeasts of which the outgrowth can be controlled Saccharomyces and Zycosaccharomyces were used.

It is further shown that the killer toxins of the present invention is active against a wide range of yeasts and fungi. Activity is shown against Brettanomyces anomalus, Brettanomyces intermedius, Candida glabrata, Debaryomyces hansenii, Dekkera anomala, Dekkera naardenensis, Hansenula arabitolgenus, Hanseniaspora uvarum, Pichia anomala, Pichia angusta, Pichia kluvveri, Saccharomyces cerevisiae, Saccharomyces exiguus, Williopsis saturnus, Zygosaccharomyces bailii, Zygosaccharomyces microellipsoides, Zygosaccharomyces rouxii, Candida boidinii, Candida etchellsii, Hanseniaspora osmophila, Pichia membraneafaciens, Rhodotorula mucilaginosa and Schizosaccharomyces pombe

The invention further discloses a DNA fragment containing the gene encoding zymocin. The DNA is isolated using standard procedures.

The present invention also discloses the DNA sequence encoding zymocin obtainable from Williopsis mrakii. It is understood that these sequences can be used to device probes suitable for the identification of zymocin encoding genes from other Hansenula and Williopsis species. After identification the detected genes can be cloned and used for protein production as examplified herein by the Williopsis mrakii gene.

The sequence encoding the zymocins from other species can be cloned in suitable expression systems as shown in the examples for the Williopsis mrakii gene.

Furthermore cloning and expression vectors and host species for obtaining zymocin are disclosed.

The present invention makes possible the cloning, overexpression and large scale production of zymocin.

The zymocin can thus be obtained in large amounts and with a high purity. The zymocin gene can be cloned in suitable expression vectors. The choice of such vectors is dependent on the host organism. The host organism is preferably chosen from among species which are not susceptible to the activity of the zymocin. Examples of such organisms are Bacilli and Aspergilli. Preferred species are Bacillus subtilis, Bacillus licheniformis, Bacillus alkaligenes, Bacillus amyloliguefaciens, Bacillus lentus and Bacillus stearothermophilus. Preferred Aspergilli are Aspergillus niger and Aspergillus nidulans.

For the majority of species indicated cloning and expression vectors are known in the art and can be used for zymocin.

This method for preparing the zymocin is especially advantages since it is known that the Williopsis mrakii produces other killer toxines as well. By heterologous cloning and expression of the zymocin this source of contamination is avoided.

The zymocin can be used without extensive purification either in the presence or in the absence of the production host. Alternatively the zymocin can be purified, methods are known in the art. In the present examples FPLC is used, HPLC is another possibility. If zymocin is to be produced on a large scale different chromatographic methods are available, including ion-exchange chromatography and gel filtration.

EXAMPLE 1 Isolation of Zymocin from Williopsis

Strains

Williopsis mrakii IFO 895, Hansenula saturnus IFO 117 and Hansenula saturnus IFO 1466 were used as the zymocin producers. Hansenula anomala IFO 569 was used as a sensitive tester strain. Both strains were obtained from the Institute for Fermentation, Osaka, Japan. All other strains were obtained from the Centraalbureau voor Schimmelcultures (CBS), Baarn, The Netherlands.

Media

YEPD-broth consisted of Bacto-yeast extract (Difco) 1%, Bacto-peptone (Difco) 2% and glucose 2% in 50 mM citrate-phosphate buffer (pH 4.8). The complex medium was used to prepare seed cultures. Bacto-yeast nitrogen base (YNB) 1%, glucose 10% in 50 mM citrate-phosphate buffer (pH 4.4) was used as minimal production medium in shake flask fermentations.

Detection of the Activity

Serial dilutions (1:1) of the culture broth were made in microtiter plates. To each well an equal volume (100 μl) of a suspension of Hansenula anomala IFO 569 (2.10⁴ -2.10⁵ cells per ml) was pipetted. The plates were incubated for 3 days at 25° C. The minimal concentration of zymocin, that inhibited the growth (MIC) of Hansenula anomala IFO 569 under these conditions, was defined to contain 1 arbitrary unit (AU) per ml.

To determine the Minimal Lethal Concentration (MLC) of zymocin, 5 μl from each well was spotted on YEPD, 2% agar plates. The plates were incubated for 2 days at 25° C.

Production

Small scale fermentations (100 ml) were carried out with Hansenula mrakii IFO 895 and both Hansenula saturnus strains, in 500 ml shake flasks with baffles. The flasks were incubated in an Incubator Shaker (New Brunswick Scientific, Edison, N.J., USA), at 250 r.p.m. at 25° C. for 72 hours.

Large scale fermentations were carried out in a 10 liter fermentor unit (L. Eschweiler & Co., Kiel, Germany). Four liter of YNB 2.5%, glucose 2.5% was inoculated with 1 liter of an overnight culture of Williopsis mrakii IFO 895 in YNB 1%, glucose 2.5%. After a batch phase of 15 hours a glucose solution (27.5%) was fed to the culture (100 gram per hour) for another 34 hours. The pH was kept at pH 5.0 using 4N NaOH/4N H₂ SO₄, the temperature was kept at 25° C. The stirring rate was 300 rpm.

Purification

Cells were harvested by centrifugation. The filtrate was filter sterilized and the zymocin was precipitates by addition of 2 volumes of methanol. The precipitate was collected by filtration over sintered stainless steel and dried under vacuum at room temperature. The precipitate was dissolved (1.7%) in 10 mM phosphate buffer (pH 3.4) and loaded on S Sepharose Fast Flow HR 16/9.4 using a BioPilot FPLC unit (Pharmacia LKB, Uppsala, Sweden). Zymocin was eluted with 1M NaCl in 10 mM phosphate buffer (pH 3.4). The fractions, that contained zymocin activity were pooled, dialysed against 10 mM phosphate buffer (pH 4.4) and stored frozen at -20° C.

Characterization of Zymocin

Stability of Zymocin at Different pH Values

Fermentation broth of Williopsis mrakii with a zymocin activity between 64 and 128 AU/ml and of both Hansenula saturnus strains (32-64 AU/ml), was adjusted to various pH values using either 1M H₃ PO₄ or NaOH. After stirring for 1 hour on a magnetic stirring device at room temperature, the solution was neutralized and the zymocin activity was determined. The results are presented in Table 1.

                  TABLE 1                                                          ______________________________________                                         Zymocin activity (AU/ml)                                                       Williopsis mrakii  Hansenula saturnus                                          pH     IFO 895         IFO 117  IFO 1466                                       ______________________________________                                          1     32-64           n.d.     n.d.                                            2     64-128          n.d.     n.d.                                           3-9    64-128          32-64    32-64                                          10     64-128          n.d.     n.d.                                           11     64-128          n.d.     n.d.                                           12     16-32           n.d.     n.d.                                           ______________________________________                                          n.d. not detected                                                        

Stability of Zymocin at High Temperatures

Fermentation broth of Williopsis mrakii (64-128 AU zymocin/ml) and of both Hansenula saturnus strains (32-64 AU/ml) was heated for 10 minutes at 80°, 100° and 120° C., respectively. The samples were cooled to room temperature and the zymocin activity was measured. The results are shown in Table 2.

                  TABLE 2                                                          ______________________________________                                                 Zymocin activity (AU/ml)                                                       Williopsis mrakii                                                                          Hansenula saturnus                                         Temperature                                                                              IFO 895       IFO 117  IFO 1466                                      ______________________________________                                         25° C.                                                                            64-128        32-64    32-64                                         80° C,                                                                            64-128        32-64    32-64                                         100° C.                                                                           64-128        16-32    16-32                                         120° C.                                                                           16-32          8-16    4-8                                           ______________________________________                                    

EXAMPLE 2 Activity of Zymocin Against Various Yeast Strains

Twenty-six strains of various yeast species were used as indicator organism in the activity tests described in example 1. The MIC and MLC-values are given in Table 3.

                                      TABLE 3                                      __________________________________________________________________________                  Williopsis mrakii                                                                             Hansenula saturnus                                              IFO 895        IFO 117   IFO 1466                                              Strain                                                                              MIC  MLC  MIC  MLC  MIC  MLC                                 Species      CBS No.                                                                             (AU/ml)                                                                             (AU/ml)                                                                             (AU/ml)                                                                             (AU/ml)                                                                             (AU/ml)                                                                             (AU/ml)                             __________________________________________________________________________     Brettanomyces anomalus                                                                      77   64   128  >512 >512 1024 1024                                Brettanomyces intermedius                                                                   1940 16   16   >512 >512 64   1024                                Candida glabrata                                                                            2663 4    4    2    4    1    2                                   Debaryomyces hansenii                                                                       4373 128  256  >512 >512 >1024                                                                               >1024                               Dekkera anomala                                                                             8138 64   128  8    8    4    4                                   Dekkera naardenensis                                                                        6115 16   32   n.d. n.d. n.d. n.d.                                Dekkera naardenensis                                                                        6041 4    8    >512 >512 1024 1024                                Hansenula arabitolgenus                                                                     7164 8    16   n.d. n.d. n.d. n.d.                                Hanseniaspora uvarum                                                                        5934 1024 1024 n.d. n.d. n.d. n.d.                                Pichia anomala                                                                              1683 16   16   16   16   8    8                                   Pichia angusta                                                                              1976 128  1024 n.d. n.d. n.d. n.d.                                Pichia kluyveri                                                                             6859 4    4    4    8    4    4                                   Saccharomyces cerevisiae                                                                    425  256  512  >512 >512 512  512                                 Saccharomyces cerevisiae                                                                    1552 32   64   16   32   16   16                                  Williopsis saturnus                                                                         7192 >1024                                                                               >1024                                                                               n.d. n.d. n.d. n.d.                                Zygosaccharomyces bailii                                                                    1097 16   16   256  256  8    8                                   Zygosaccharomyces                                                                           427  16   32   32   32   16   16                                  microellipsoides                                                               Zygosaccharomyces rouxii                                                                    711  512  1024 256  256  256  256                                 Zygosaccharomyces rouxii                                                                    4564 256  512  n.d. n.d. n.d. n.d.                                Candida boidinii                                                                            6368 inhibited                                                                           inhibited                                                                           n.d. n.d. n.d. n.d.                                Candida etchelsii                                                                           2987 inhibited                                                                           inhibited                                                                           n.d. n.d. n.d. n.d.                                Hanseniaspora osmophila                                                                     4266 inhibited                                                                           inhibited                                                                           n.d. n.d. n.d. n.d.                                Pichia membraneafaciens                                                                     5756 inhibited                                                                           inhibited                                                                           n.d. n.d. n.d. n.d.                                Rhodotorula mucilaginosa                                                                    334  inhibited                                                                           inhibited                                                                           n.d. n.d. n.d. n.d.                                Schizosaccharomyces pombe                                                                   1042 inhibited                                                                           inhibited                                                                           n.d. n.d. n.d. n.d.                                __________________________________________________________________________

EXAMPLE 3

Activity of Zymocin Against Yeasts in Carbonated Beverages

Two yeast strains: Saccharomyces cerevisiae CBS 425 and Zygosaccharomyces rouxii CBS 711, were grown in YEPD as described in Example 1. Suspensions (2.10⁵ cells/ml) were made in various carbonated beverages. The same beverages were used to make serial dilutions (1:1) of zymocin (2048-2 AU/ml). Aliquot of 100 μg of the zymocin solution were brought into the wells of a microtiter plate. An equal volume of the yeast suspension in the carbonated beverages was pipetted into the wells and the MIC and MLC values were determined as described in Example 1. The results are shown in Table 4. The zymocin shows a high activity especially against S. cerevisiae.

                  TABLE 4                                                          ______________________________________                                                  S. cerevisiae CBS 425                                                                       Z. rouxii CBS 711                                                   MIC      MLC       MIC    MLC                                       Product    (AU/ml)  (AU/ml)   (AU/ml)                                                                               (AU/ml)                                   ______________________________________                                         Apple-juice                                                                               128      256       128    256                                       Pear-juice 128      256       128    256                                       Orange-juice                                                                              256      512       128    256                                       Orange-juice                                                                              128      n.d.      128    n.d.                                      II                                                                             Grape-juice                                                                               >512     >512      512    512                                       Vegetable- 512      1024      256    >1024                                     juice                                                                          Bron Orange*                                                                              16       16        256    256                                       Jaffa Drink*                                                                              16       16        128    128                                       Stender B- 16       16        64     64                                        ier**                                                                          Control: YEPD                                                                             256      256       512    1024                                      ______________________________________                                          *Carbonated lemonades                                                          **Nonalcoholic beer                                                            n.d. Not detected                                                              Bron Orange ™, Jaffa Drink ™ and Stender Bier ™ are Trademarks. 

EXAMPLE 4 Activity of Zymocin Against Zygosaccharomyces bailli in Black Currant Drink

Zymocin is tested in black currant drink, Zygosaccharomyces bailii CBS 1097 was grown for 24 hours at 30° C. in 25 ml BHI medium in a 100 ml shake flasks at 250 rpm in a shaking incubator (New Brunswick Scientific, Edison, N.J., USA). Serial dilutions of Zymocin in 25 ml black currant drink were made (0 to 250 AU/ml) and pipetted in 100 ml erlenmeyers. The Zymocin solutions were inoculated with the test strain in a concentration of 10³ cells per ml. The flasks were incubated at 30° C. After 1 day, the number of viable cells was determined by total plate counting on YEPD-agar.

                  TABLE 5                                                          ______________________________________                                         Zymocin conc. (U/ml)                                                                          Number of viable cells/ml                                       ______________________________________                                          0             >100.000                                                         10            6000                                                             50            3015                                                            125            1770                                                            250            1040                                                            ______________________________________                                    

EXAMPLE 5 Activity of Various Zymocin Against Yeasts Strains in Non-alcoholic Beer

Four yeast strains: Saccharomyces cerevisiae CBS 425, Saccharomyces cerevisiae CBS 1552, Saccharomyces exiguus CBS 6946 and Zygosaccharomyces bailii CBS 1097 were tested for their sensitivity to variuos zymocins in non-alcoholic beer. The MIC and MLC values were determined the results are presented in Table 6.

    ______________________________________                                                    Zymocin activity (AU/ml)                                                       W. mrakii                                                                              Hansenula saturnus                                                     IFO 895 IFO 117    IFO 1466                                         Strains      MIC    MLC    MIC  MLC   MIC  MLC                                 ______________________________________                                         S. cerevisiae CBS 425                                                                       8      16     32   64    16   22                                  S. cerevisiae CBS 1552                                                                      2      4      8    8     4    8                                   S. exiquus CBS 6946                                                                         8      8      8    8     8    8                                   Z. bailii CBS 1097                                                                          1      1      4    4     0.5  0.5                                 ______________________________________                                    

EXAMPLE 6 Activity of Various Zymocins Against Yeasts in Cerise™ and 7-Up™

Two yeast strains: Zygosaccharomyces bailii CBS 1097 and Saccharomyces exiguus CBS 6946 were grown in Cerise™ and 7-Up™, respectively. The MIC values were determined as described in Example 3. The results are presented in Table 7.

                  TABLE 7                                                          ______________________________________                                                        Zymocin activity (AU/ml)                                                       W. mrakii                                                                              Hansenula saturnus                                                           IFO 895   IFO 117                                                                              IFO 1466                                  Drink  Strain        MIC       MIC   MIC                                       ______________________________________                                         Cerise ™                                                                           Z. bailii CBS 1097                                                                           256       256   256                                       7-Up ™                                                                             S. exiguus CBS 6946                                                                          128       128   128                                       ______________________________________                                    

EXAMPLE 7 Cloning of the Zymocin Gene of Williopsis mrakii

The amino acid sequence of Williopsis mrakii Zymocin is published earlier by Yamamoto, T., FEBS Letters 195 (1986), 253-257. Using these data a set of degenerated oligonucleotides was derived which coded for a chosen string of amino acids. The following oligonucleotides were synthesized:

    __________________________________________________________________________       7  8  9  10 11 12                                                            5'-                                                                              Met                                                                               Cys                                                                               Lys                                                                               Asn                                                                               Cys                                                                               Asp - 3'                                                                           (Seq. ID No: 1 and Seq ID No: 2)                            ATG                                                                               TGT                                                                               AAA                                                                               AAT                                                                               TGT                                                                               GAT                                                                  C                                                                                 G                                                                                 C                                                                                 C                                                                80 79 78 77 76 75 74 73                                                      5'-                                                                              Tyr                                                                               Phe                                                                               GLy                                                                               Thr                                                                               Asn                                                                               Ile                                                                               Asn                                                                               Cys - 3'                                                                           (Seq. ID                                                                       No: 3 and 4)                                          ATA                                                                               AAA                                                                               ACC                                                                               AGT                                                                               ATT                                                                               AAT                                                                               ATT                                                                               ACA                                                       G  G  T  T  G  T  G  G                                                               C  C     G                                                                     G  G                                                                   __________________________________________________________________________

A PCR experiment was carried out using the given oligo nucleotides as primers and chromosomal DNA of Williopsis mrakii IFO 895 as a template. In doing the PCR experiment a DNA fragment from Williopsis mrakii was amplified, with a length of 222 bp as expected. This fragment was used as a hybridization probe for a Southern blots of chromosomal Williopsis mrakii DNA, digested with various restriction enzymes. Positive hybridization signals were found with a Pst I fragments of 5 kb. Subsequently Pst I digested chromosomal DNA of Williopsis mrakii was separated by agarose gel electrophoresis on preparative scale. Fragments of 5±0.5 kb were isolated from the gel and ligated with the commercial available cloning vector pUC18 (Appligene, Strassbourg, France). The ligation mixtures were transformed to Escherichia coli XL1-Blue (Stratagene, La Jolla, Calif., USA). The 222 bp PCR fragment was used as a probe in a colony hybridization experiment to select for clones giving a positive hybridization signal. Two types of transformants were obtained, carrying pUC18 derived plasmids with the same 5 kb PstI fragment in 2 different orientations. The plasmids were called pUC18K1 and PUC18K2.

A part of the 5 kb fragment was sequenced using the oligo nucleotides mentioned above. The nucleotide sequence was translated in 3 different reading frames. One of these reading frames showed an amino acid sequence that contained the identical sequence of 88 amino acid, published before. Therefore we conclude we cloned the gene coding for the zymocin of Williopsis mrakii. The nucleotide sequence and the derived amino acid sequence of the zymocin gene is shown in FIG. 1 (Seq. ID No: 5 and 6)

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 6                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 6 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (v) FRAGMENT TYPE: internal                                                    (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Williopsis mrakii                                                (B) STRAIN: IFO 895                                                            (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: peptide1                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        MetCysLeuAsnCysAsp                                                             15                                                                             (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Williopsis mrakii                                                (B) STRAIN: IFO 895                                                            (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: oligo1                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        ATGTGYAARAAYTGYGAT18                                                           (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 8 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (v) FRAGMENT TYPE: internal                                                    (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Williopsis mrakii                                                (B) STRAIN: IFO 895                                                            (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: peptide2                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        TyrPheGlyThrAsnIleAsnCys                                                       15                                                                             (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 24 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: YES                                                           (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Williopsis mrakii                                                (B) STRAIN: IFO 895                                                            (vii) IMMEDIATE SOURCE:                                                        (B) CLONE: oligo2                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        RTARAANCCNGTRTTDATRTTRCA24                                                     (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1250 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                            (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Williopsis mrakii                                                (B) STRAIN: IFO 895                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 424..801                                                         (D) OTHER INFORMATION: /codon.sub.-- start= 424                                /product= "Zymocin"                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        TTAGCATGATTGCCTATTGGATCGGTGCTTCACAGTAATTGTTGTTCAAACTGATCAAGA60                 AGCACAAGGAAGGGTGCGCTCAATTGGTACTTTCGCAGCAAGATCAGGAAGCAGGTCGCA120                GACTTCGACCACTTGCGGTCTCGAGAAGTTGTCAGCGAAGAAAGAAAACAGAGTTCAGCA180                ACTTGTCGGATTAGGAAAACTGTATGCAATGCAATGCGCAAAAACGCTGTCTGATCACTG240                GGTGTTTATCCATCAGAGAGTGAAGCACGCGGAGCTGCCACAAACCAACTTCGAAGATTT300                TCGTTAGATGGGTATATAAAGACTTCCACTTCCCTGACAAGTCCCATACACTGTCTTCCC360                ACATCTTTCCCCTTGAATCACGAAAAAAGTCAGGAACTTTTGTACTTGACATTTCCGCCC420                AACATGAAATTTTCCTTCGTTTACGGTTTGACAGGCTTCTTAGCAGCA468                            MetLysPheSerPheValTyrGlyLeuThrGlyPheLeuAlaAla                                  151015                                                                         ACCTCATCAGCTTTGCCTTCCGAAATCTTATCAACTGGCTATGAAAGA516                            ThrSerSerAlaLeuProSerGluIleLeuSerThrGlyTyrGluArg                               202530                                                                         AGTGCATTGGAGAAGCGTGGTGATGGATACCTGATTATGTGCAAAAAC564                            SerAlaLeuGluLysArgGlyAspGlyTyrLeuIleMetCysLysAsn                               354045                                                                         TGTGACCCAAACACGGGTAGCTGTGATTGGAAGCAGAACTGGAATACT612                            CysAspProAsnThrGlySerCysAspTrpLysGlnAsnTrpAsnThr                               505560                                                                         TGTGTAGGCATAGGAGCTAATGTCCACTGGATGGTTACAGGCGGCAGC660                            CysValGlyIleGlyAlaAsnValHisTrpMetValThrGlyGlySer                               657075                                                                         ACTGATGGGAAGCAAGGGTGTGCTACAATCTGGGAAGGCTCAGGATGT708                            ThrAspGlyLysGlnGlyCysAlaThrIleTrpGluGlySerGlyCys                               80859095                                                                       GTGGGTAGATCAACCACAATGTGTTGTCCGGCCAATACTTGTTGCAAC756                            ValGlyArgSerThrThrMetCysCysProAlaAsnThrCysCysAsn                               100105110                                                                      ATCAACACAGGGTTCTACATTCGCTCTTACAGACGTGTGGAATAG801                               IleAsnThrGlyPheTyrIleArgSerTyrArgArgValGlu*                                    115120125                                                                      GTGATTAACTATATCAACCGTAATGGAAAATTCGTGAACCGAAATTTCATTTTTGAGGAT861                ACCACACCAACTTGCGCTCAGATCCGTCACGTTATATATTTCTATAATTTACGCTTTAAT921                AGGTGAATGTACTATGAAGGCCTCTTTAGAAATTTATACCTCTTTTGATTATAGTGGCAC981                TATGATTATATAGTGCGTGTGATTGCTAGAGAAAATTTCAGAAAACTTCCGTATCGTCTG1041               CACTGACATTGCTCAAGCACCAAAAAAGCAGTCTCCTGATATGTCACTTAAGATCAAGAA1101               TAGATGTTTAGATGTAGCTAGTGTTTATCTGACAATAAAAGGACTCCTAAGGTACAAACG1161               GCTGAGAGGCAAAGACTTTGAGGAATTTTGCTCAAGATTCTGTGATTCAGCCTTTACCAG1221               CGACCTATCCATGCACGAAGTACTCTCTA1250                                              (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 125 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        MetLysPheSerPheValTyrGlyLeuThrGlyPheLeuAlaAlaThr                               151015                                                                         SerSerAlaLeuProSerGluIleLeuSerThrGlyTyrGluArgSer                               202530                                                                         AlaLeuGluLysArgGlyAspGlyTyrLeuIleMetCysLysAsnCys                               354045                                                                         AspProAsnThrGlySerCysAspTrpLysGlnAsnTrpAsnThrCys                               505560                                                                         ValGlyIleGlyAlaAsnValHisTrpMetValThrGlyGlySerThr                               65707580                                                                       AspGlyLysGlnGlyCysAlaThrIleTrpGluGlySerGlyCysVal                               859095                                                                         GlyArgSerThrThrMetCysCysProAlaAsnThrCysCysAsnIle                               100105110                                                                      AsnThrGlyPheTyrIleArgSerTyrArgArgValGlu                                        115120125                                                                      __________________________________________________________________________ 

We claim:
 1. A method for preserving non-alcoholic, non-carbonated beverages comprising adding an effective amount of a yeast zymocin to said beverage.
 2. A method according to claim 1 wherein said non-carbonated beverage contains fruit, fruit juice or a fruit extract.
 3. A method according to claim 2 wherein the beverage is selected from the group consisting of apple juice, orange juice, pear juice and black currant drink.
 4. A method according to claim 1 wherein said zymocin is obtainable from a yeast strain selected from a genus consisting of Hansenula and Williopsis.
 5. A method according to claim 1 wherein said zymocin is added to achieve a final concentration of 128 to 512 AU/ml.
 6. A method according to claim 5 wherein said zymocin is produced from an organism that has been altered to contain a DNA molecule that encodes said zymocin.
 7. A method according to claim 6 wherein said DNA molecule encodes a zymocin having the amino acid sequence depicted in Sequence ID No:5.
 8. A non-alcoholic, non-carbonated beverage having been preserved by the method of claim
 5. 